Serovar-dependent differences in Hfq-regulated phenotypes in actinobacillus pleuropneumoniae

The RNA chaperone Hfq regulates diverse processes in numerous bacteria. In this study, we compared phenotypes (growth rate, adherence, response to different stress conditions, and virulence in Galleria mellonella) of wild-type (WT) and isogenic hfq mutants of three serovars (1, 8 and 15) of the porcine pathogen A. pleuropneumoniae. Similar growth in rich broth was seen for all strains except Ap1∆hfq, which showed slightly reduced growth throughout the 24 hour time course, and the complemented Ap8∆hfqC mutant had a prolonged lag phase. Differences were seen between the three serovar WT strains regarding adherence, stress response and virulence in G. mellonella, and deletion of hfq affected some, but not all of these phenotypes, depending on serovar. Complementation by expression of cloned hfq from an endogenous promoter only restored some WT phenotypes, indicating that complex regulatory networks may be involved, and that levels of Hfq may be as important as presence/absence of the protein regarding its contribution to gene regulation. Our results support that Hfq is a pleiotropic global regulator in A. pleuropneumoniae, but serovar-related differences exist. These results highlight the importance of testing multiple strains/serovars within a given species when determining contributions of global regulators, such as Hfq, to expression of complex phenotypes

Use of Galleria mellonella as a model of infection and study of factors related to the virulence of Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae é o agente etiológico da pleuropneumonia suína, uma severa enfermidade que acomete suínos de todas as idades, gerando perdas econômicas significativas para a suinocultura mundial. Embora os 15 sorotipos conhecidos dessa bactéria possam causar a doença, existem diferenças marcantes de virulência entre eles. A virulência de A. pleuropneumoniae é multifatorial e está relacionada à composição e estrutura de polissacarídeos da cápsula, LPS, e toxinas da família RTX, além desses fatores, a aderência em forma de biofilme e a resistência a agentes antimicrobianos podem ser determinantes para virulência. Este trabalho estabeleceu um modelo de infecção alternativo para o estudo de A. pleuropneumoniae, utilizando larvas de Galleria mellonella e, posteriormente esse modelo foi usado para investigar a virulência de isolados clínicos de A. pleuropneumoniae sorotipo 8. Os mesmos isolados foram avaliados quanto ao potencial de formação de biofilme e resistência a antimicrobianos comumente empregados em campo. A partir dessas informações, isolados clínicos com diferenças significativas na virulência, no potencial de formação de biofilme e no perfil de resistência foram selecionados para o sequenciamento genômico. Os resultados mostraram que o modelo de infecção A. pleuropneumoniae – G. mellonella é capaz de diferenciar níveis de virulência de isolados clínicos de mesmo sorotipo, além de permitir a avaliação da eficiência de agentes antimicrobianos contra este patógeno. O modelo também mostrou eficiência para diferenciar virulência entre linhagens selvagem e mutante da mesma bactéria. Uma análise de correlação entre os dados de virulência, formação de biofilme e resistência a antimicrobianos permitiu que seis isolados fossem selecionados para o sequenciamento. Com a montagem e anotação foi possível verificar que os genomas de A. pleuropneumoniae sorotipo 8 apresentam tamanho de 2,2 ± 0,004 Mpb, com o conteúdo GC de 40,33% ± 0,263 e regiões codificadoras com uma média de tamanho de 817,3 ± 6,8 pb. As regiões codificadoras correspondem a 89,05% ± 0,13 do genoma, das quais a maior parte foi anotada como genes funcionais, o que permitirá a realização de estudos comparativos. Estes genomas apresentam em média 79,5 ± 24,05 genes exclusivos, revelando a alta variabilidade genética dessa espécie, que pode estar relacionada com a variação da virulência entre os isolados estudados.Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a severe disease that affects pigs of all ages, causing significant economic losses to the swine industry worldwide. Although the 15 serotypes of this bacterium are known to cause the disease, there are marked differences in virulence between them. A. pleuropneumoniae virulence is multifactorial and involves capsular polysaccharides, LPS, and toxins of the RTX family. In addition to these factors, the adhesion in biofilm form and resistance to antimicrobial agents may be determinant for virulence. This work has established an alternative infection model for the study of A. pleuropneumoniae, using larvae of Galleria mellonella and this model was subsequently used to investigate the virulence of clinical isolates of A. pleuropneumoniae serotype 8. The same isolates were evaluated for biofilm formation potential and resistance to antimicrobials commonly used in the field. From this information, clinical isolates with significant differences in virulence, biofilm formation potential and resistance profile were selected for genomic sequencing. Results show that the A. pleuropneumoniae - G. mellonella infection model is capable of differentiating levels of virulence of clinical isolates of the same serotype. Furthermore, it can be used to evaluate the effectiveness of antimicrobial agents against this pathogen. The model also showed efficiency to differentiate the virulence between wild and mutant strains of the same bacteria. A correlation analysis between the virulence data, biofilm formation and antibiotic resistance allowed six isolates to be selected for genome sequencing. With the assembly and annotation, we found that the genomes of A. pleuropneumoniae serotype 8 present size of 2.2 ± 0.004 Mpb, with GC content of 40.33% ± 0.263 and coding regions with an average size of 817.3 ± 6.8 bp. The coding regions correspond to 89.05 ± 0.13% of the genome, most of which was recorded as functional genes, enabling the comparative studies with these genomes. These genomes have 79.5 ± 24.05 exclusive proteins, revealing the high genetic variability of the species, which may be related to the variation in virulence between these isolates.Conselho Nacional de Desenvolvimento Científico e Tecnológic

Functional study of a gene that encodes a MFS membrane transporter in Colletotrichum lindemuthianum

O fungo Colletotrichum lindemuthianum é o agente causal da antracnose do feijoeiro comum. Este fungo, assim como outros fungos fitopatógenos estão constantemente expostos a uma grande variedade de compostos tóxicos provenientes de várias fontes, o que torna imprescindível para estes o desenvolvimento de mecanismos de proteção contra estes produtos. Uma dessas estratégias está relacionada com a presença de proteínas transportadoras de membrana, como as pertencentes à Principal Superfamília Facilitadora (MFS), que podem fornecer aos fungos proteção contra compostos tóxicos evitando ou minimizando a ação destes, sendo em sua maioria essenciais para a manutenção da viabilidade celular. Este trabalho teve como objetivo inativar o gene mfs1 que codifica para um transportador de membrana da família MFS e investigar as alterações fenotípicas ocasionadas em um mutante C. lindemuthianum isolado LV49 (raça 89) para este gene. Para a obtenção do mutante foi necessário confirmar que o gene mfs1 encontrava-se presente em cópia única no genoma de C. lindemuthianum. O gene mfs1 pode estar organizado em um conjunto de genes com funções relacionadas, uma vez que downstream à região 3’ deste foi identificada uma segunda janela aberta de leitura correspondente a uma proteína da superfamília de fatores de transcrição contendo o domínio Zn2-Cys6, identificado como clft1. A análise do promotor do gene mfs1 revelou um putativo cis elemento de reconhecimento por proteínas desta família, o que sugere que esta proteína possa estar relacionada à regulação da expressão de mfs1. A técnica de Split- Marker mostrou-se eficiente na inativação do gene mfs1 de C. lindemuthianum, possibilitando o estudo da função do gene mfs1, em um mutante com integração específica e livre de integrações ectópicas. O mutante Δmfs1 não mostrou diferenças no perfil de sensibilidade a drogas comumente empregadas no controle da antracnose e em relação à patogenicidade, o mutante induziu mais precocemente os sintomas em folhas de feijoeiro susceptível, evidenciando uma situação de estresse decorrente da ausência do produto gênico. Foi observado também que o gene mfs1 exerce um papel primordial na manutenção da viabilidade celular de C. lindemuthianum, fato este confirmado pela conidiação alterada e pela confirmação de que este gene codifica para um transportador de membrana específico no transporte de hexoses, especificamente glicose, manose e frutose, uma vez que o mutante Δmfs1 mostrou crescimento reduzido quando cultivado em meios contendo apenas glicose, manose e frutose como fontes de carbono. A análise filogenética da proteína Mfs1 associada aos outros resultados obtidos nos sugere que este transportador é um membro da família SP, e que as proteínas MFS estão fortemente relacionadas com o tipo de substância que é transportada. Estudos de natureza básica sobre transportadores MFS são importantes para ampliar os conhecimentos sobre estas proteínas e a viabilidade celular em C. lindemuthianum.Colletotrichum lindemuthianum is the causal agent of common bean antracnose. This fungus, like other phytopatogens, is constantly exposed to several toxic compounds from many sources, what makes indispensable the development of protection strategies against these products. One of these strategies is related to membrane transporters proteins like the Major Facilitator Superfamily (MFS), that could provide protection against toxic compounds or minimizing its action, being essential for the fungal cellular viability maintenance. In this context, this work aimed to inactivate the mfs1 gene encoding a MFS membrane transporter and investigate the phenotypic alterations entailed in an isolate C. lindemuthianum mutant LV49 (race 89) for this gene. To obtain the mutant, it was necessary to confirm if mfs1 gene was organized as a single copy in the C. lindemuthianum genome. The mfs1 gene can be organized in a cluster in view that a second open reading frame, which corresponds to a transcription factor superfamily containing a Zn2-Cys6 domain, identified as clft1, was observed in 3` downstream region of this gene. The mfs1 promoter analysis revealed a putative mfs1 element that is recognized by proteins of this family, what suggests that this protein could be related to the mfs1 expression regulation. The Split-Marker technique proved to be efficient in C. lindemuthianum mfs1 gene inactivation enabling the study of mfs1 function in a mutant by specific integrations without ectopic integrations. The Δmfs1 mutant showed no differences in drug sensibility profile when commonly drugs employed in antracnose control was used and in relation to pathogenicity, the mutant symptoms started earlier on susceptible bean leaves, showing a stress situation due to the genic product absence. It was also observed that mfs1 presents a primordial role in C. lindemuthianum cellular viability maintenance, what was confirmed by the altered conidiation observed, confirming that this gene encodes for a specific hexose membrane transporter, specifically carbon sources like glucose, mannose and fructose. The protein Mfs1 phylogenetic analysis allow us to conclude that this transporter is a SP family member and the MFS proteins are strongly related with the transported substance. Studies conducted with MFS transporters are important to broaden the knowledge of these proteins and to understand the cell viability in C. lindemuthianum.Conselho Nacional de Desenvolvimento Científico e Tecnológic

Editorial

Editorial A atual conjuntura sanitária que o Brasil e o mundo vivem em decorrência da pandemia da COVID-19 (Coronavírus disease - 2019), colocou em evidência a necessidade de ampliar os conhecimentos e os debates sobre as viroses zoonóticas emergentes e seus impactos sobre a saúde pública. No entanto, para entender o que são e o que representam as viroses zoonóticas emergentes, é necessário destacar que essas são extremamente dinâmicas, e que estão relacionadas com contextos sociais e ambientais que se diferem ao longo do tempo e também do lugar

A emergência de um novo coronavírus zoonótico: SARS-CoV-2 e a pandemia da COVID-19

Os coronavírus impõem uma ameaça contínua aos seres humanos e à saúde pública mundial, pois emergem inesperadamente, se espalham facilmente e levam a consequências catastróficas. O século XXI está marcado pela ocorrência de três significativas viroses zoonóticas causadas pelos coronavírus SARS-CoV, MERS-CoV e SARS-CoV-2. Atualmente estamos vivenciando a pandemia de COVID-19, uma grave doença respiratória causada por SARS-CoV-2. Os esforços para reconstituir a história epidemiológica da COVID-19 são intensos e necessários para que o problema atual não se repita em um futuro próximo. As experiências prévias com SARS-CoV e MERS-CoV foram valiosas para direcionar as medidas de controle de SARS-CoV-2. No entanto, até o momento não existe um tratamento eficaz ou uma vacina contra a COVID-19, e simples práticas preventivas combatem a disseminação do vírus. Diante disso, este artigo resume o atual conhecimento sobre os aspectos biológicos, epidemiológicos, clínicos e terapêuticos relacionados a SARS-CoV-2 e a COVID-19

Antimicrobial resistance, biofilm formation and virulence reveal Actinobacillus pleuropneumoniae strains' pathogenicity complexity

Porcine pleuropneumonia is an important cause of lowered productivity and economic loss in the pig industry worldwide, associated primarily with Actinobacillus pleuropneumoniae infection. Its colonization and persistence within the upper respiratory tract of affected pigs depends upon interactions between a number of genetically controlled virulence factors, such as pore-forming repeats-in-toxin exoproteins, biofilm formation, and antimicrobial resistance. This study investigated correlations between biofilm-forming capacity, antimicrobial resistance, and virulence of A. pleuropneumoniae obtained from clinical outbreaks of disease, using a Galleria mellonella alternative infection model. Results suggest that virulence is diverse amongst the 21 strains of A. pleuropneumoniae examined and biofilm formation correlated with genetic control of antimicrobial resistance

Clonal dissemination of vancomycin-resistant Enterococcus faecium ST412 in a Brazilian region

Vancomycin-resistant Enterococcus faecium (VREfm) has emerged as an important global nosocomial pathogen, and this trend is associated with the spread of high-risk clones. Here, we determined the genetic and phenotypic features of 93 VREfm isolates that were obtained from patients in 13 hospitals in Vitória, Espírito Santo, Brazil, during 2012–2013. All the isolates were vancomycin-resistant and harbored the vanA gene. Only 6 (6.5%) of the VREfm isolates showed the ability to form biofilm. The 93 isolates analyzed belong to a single pulsed-field gel electrophoresis lineage and presented six subtypes. MLST genotyping showed that all VREfm belonged to ST412 (the high-risk clone, hospital-adapted). The present study describes the dissemination of ST412 clone in the local hospitals. The clonal spread of these ST412 isolates in the area we analyzed as well as other hospitals in southeastern Brazil supports the importance of identifying and controlling the presence of these microorganisms in health care-related services. Keywords: Enterococcus faecium, VRE, ST412, Biofil

Beginning to understand the role of sugar carriers in Colletotrichum lindemuthianum: the function of the gene mfs1

Fungi of the Colletotrichum genus are among the most prominent phytopathogens that cause diseases with a considerable economic impact, such as anthracnose. The hemibiotrophic fungus Colletotrichum lindemuthianum (teleomorph Glomerella cingulata f. sp. phaseoli) is the causal agent of the anthracnose of the common bean; and similarly to other phytopathogens, it uses multiple strategies to gain access to different carbon sources from its host. In this study, we examine mfs1, a newly identified C. lindemuthianum hexose transporter. The mfs1 gene is expressed only during the necrotrophic phase of the fungus’ interaction within the plant and allows it to utilize the available sugars during this phase. The deletion of mfs1 gene resulted in differential growth of the fungus in a medium that contained glucose, mannose or fructose as the only carbon source. This study is the first to describe a hexose transporter in the hemibiotrophic pathogen C. lindemuthianum and to demonstrate the central role of this protein in capturing carbon sources during the necrotrophic development of the plant/pathogen interaction

Differential cellular immune response of Galleria mellonella to Actinobacillus pleuropneumoniae

In the present work, we have investigate the cellular immune response of Galleria mellonella larvae against three strains of the gram-negative bacterium Actinobacillus pleuropneumoniae: low-virulence (780), high-virulence (1022) and the serotype 8 reference strain (R8). Prohemocytes, plasmatocytes, granulocytes, oenocytoids and spherulocytes were distinguished according to their size and morphology, their molecular markers and dye-staining properties and their role in the immune response. Total hemocyte count, differential hemocyte count, lysosome activity, autophagic response, cell viability and caspase-3 activation were determined in circulating hemocytes of naive and infected larvae. The presence of the autophagosome protein LC3 A/B within the circulating hemocytes of G. mellonella was dependent on and related to the infecting A. pleuropneumoniae strain and duration of infection. Hemocytes treated with the high-virulence strain expressed higher levels of LC3 A/B, whereas treatment with the low-virulence strain induced lower expression levels of this protein in the cells. Moreover, our results showed that apoptosis in circulating hemocytes of G. mellonella larvae after exposure to virulent bacterial strains occurred simultaneously with excessive cell death response induced by stress and subsequent caspase-3 activation

Draft Genome Sequences of Six Actinobacillus pleuropneumoniae Serotype 8 Brazilian Clinical Isolates: Insight into New Applications

Actinobacillus pleuropneumoniae is the causative agent of swine pleuropneumonia, a highly contagious disease associated with pigs of all ages that results in severe economic losses to the industry. Here, we report for the first time six genome sequences of A. pleuropneumoniae clinical isolates of serotype 8, found worldwide